Impact of interval progression before autologous stem cell transplant in patients with multiple myeloma

In transplant-eligible patients who undergo upfront autologous stem cell transplant (ASCT) for multiple myeloma (MM), standard practice is to treat with six to eight cycles of induction therapy followed by high-dose chemotherapy with ASCT. A gap between the end of induction and the day of ASCT exists to allow stem cell mobilization and collection. Despite attempts to limit the length of this interval, we noticed that some patients experience interval progression (IP) of disease between the end of induction therapy and the day of ASCT. We analyzed 408 MM patients who underwent ASCT between 2011 and 2016. The median length of the interval between end of induction and ASCT was 38 days. We observed that 26% of patients in the entire cohort and 23.6% of patients who received induction with bortezomib-lenalidomide-dexamethasone (VRD) experienced IP. These patients deepened their responses with ASCT, independently of induction regimen. In the entire cohort, IP was significantly associated with shorter PFS in the univariable analysis (Hazard Ratio, HR = 1.37, P = 0.022) but not in the multivariable analysis (HR = 1.14, P = 0.44). However, analyzing only patients who received VRD as induction, progression-free survival (PFS) remained inferior in both the univariable (HR = 2.02; P = 0.002) and the multivariable analyses (HR = 1.96; P = 0.01). T cells and natural killer (NK) cells are increasingly studied targets of immunomodulatory therapy, as immune dysfunction is known to occur in patients with MM. Peripheral blood from 35 MM patients were analyzed. At time of ASCT, patients with IP had significantly increased percentages of CD3+CD8+CD57+ CD28- (P = 0.05) and CD3+CD4+LAG3+ (P = 0.0022) T-cells, as well as less CD56bright and CD56dim NK cells bearing activated markers such as CD69, NKG2D, and CD226. These data suggest that IP can impact the length of response to ASCT; therefore, further studies on the management of these patients are needed.


Introduction
Multiple myeloma (MM) is characterized by the abnormal growth of monoclonal plasma cells in the bone marrow, leading to bone lesions and kidney injury. MM cancer cells produce either a complete monoclonal (M) protein or free kappa or lambda light chains (FLC), which can be measured and used as markers of disease to evaluate responses to therapy and progression. Uniform response and relapse criteria have been developed by the International Myeloma Working Group (IMWG) to categorize patient responses to treatment as complete response (CR), very good partial response (VGPR), partial response (PR), minimal response (MR), and stable disease (SD) based on the percent reduction in M protein or FLC (1). Conversely, disease progression is defined as an increase of more than 25% from lowest response value or difference between involved and uninvolved FLC. More recently, the introduction of minimal residual disease (MRD) evaluation confirms the importance of achieving a deep response to therapy, even in patients in CR, to improve survival (2).
Induction therapy followed by high dose chemotherapy with autologous stem cell transplant rescue (HDT/ASCT) is still the backbone of MM treatment, providing progression-free survival (PFS) benefits compared to induction therapy alone (3). The current treatment timeline involves 4-6 cycles of induction therapy, stem cell mobilization and collection, followed by highdose conditioning chemotherapy and stem cell rescue (Supplementary Figure S1A). Stem cell collection usually results in a gap between the end of induction therapy and HDT/ASCT, during which patients do not receive disease-directed therapy. During this time interval, some patients experience interval progression (IP) of their MM, often losing part of the initial obtained response. The association between a CR after HDT/ ASCT and long-term survival has been well studied, with most retrospective analyses finding a significant association between CR and improved progression-free survival (PFS), overall survival (OS), or both (4)(5)(6)(7). The relationship between pre-HDT/ASCT CR and survival is less clear; some data suggest a pre-HDT/ASCT CR is a prognostic factor for long-term survival (8), while other studies found a survival difference that was trending towards significance only with longer follow-up (9,10). Currently no data are available regarding the post-HDT/ASCT survival outcomes associated with IP between end of induction and the day of transplant. Moreover, studies have shown that patients gain benefit from HDT/ASCT even if CR is not achieved pre-transplant (11), and thus many institutions proceed with HDT/ASCT after 4-6 cycles of induction therapy, regardless of the post-induction response.
The immune system is known to play an important role in intrinsic anti-tumoral immunity. Prior studies have demonstrated less robust immune cells in MM patients, reporting higher percentages of regulatory T cells (Treg) (12), terminallydifferentiated, senescent T cells (CD28 -CD57 + ), and exhausted T cells (lymphocyte activation gene-3 (LAG3), programmed cell death-1 (PD-1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)) (13, 14). Similarly, fewer natural killer (NK) cell activating receptors, such as Natural Killer group 2D (NKG2D), CD16, and NKp44 were found in MM patients (15). Although these data demonstrate differences in immune composition between healthy individuals and MM patients, no studies known to us have been done examining the variability in immune subpopulation among patients who experience progression of disease before ASCT. In this paper, we evaluate post-induction, pre-and post-transplant responses in patients who underwent upfront ASCT with a gap of less than 100 days from the end of induction. We specifically investigate the percentage and disease characteristics of patients who develop IP before ASCT and the relationship with post-transplant responses, PFS, and OS from day of ASCT. We also report the phenotype of their T and NK cell population prior to ASCT.

Patient selection
The patients described in this article were enrolled in a retrospective single-center study, approved by the Ohio State University Institutional Review Board (2021C0101) after providing written informed consent for the Ohio State University MM registry (OSU-10115) in accordance with the Declaration of Helsinki. A retrospective analysis of 482 patients who underwent ASCT at The Ohio State University Wexner Medical Center between 2011 and 2016 was completed.

Clinical assessment
MM labs, including protein electrophoresis with immunofixation and FLC, were obtained at diagnosis, end of induction, and Day -2 of ASCT (Supplementary Figure S1A). IP was defined as a 25% increase in the index protein between the end of induction and Day -2 of ASCT. To exclude trivial changes in the M-protein or in the KLC/LLC, patients needed to have more than 100 mg/dL of M-protein or more than 100 mg/L of free light chains in the presence of stable renal function. Treatment response was defined by the IMWG criteria at the end of induction therapy (postinduction), at Day -2 of ASCT (pre-transplant), and 30-60 days post-ASCT (post-transplant). Fluorescence In Situ Hybridization (FISH) studies were performed at diagnosis. High-risk cytogenetic abnormalities (HRCA) were considered the presence of gain/ amplification of chromosome arm 1q21 (1q21+), t(4;14), t (14;16), and deletion of chromosome 17p [del(17p)]. When two or three of these HRCAs were detected, patients were considered to have double-hit or triple-hit MM, respectively (16).

Peripheral blood sample collection and cell isolation
Peripheral blood (PB) samples from 35 patients previously collected on Day -2 of ASCT were available for analysis. PB samples from 7 healthy donors (HD) were purchased from Versiti, Inc. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation over Ficoll-Paque.

Flow cytometry staining and analysis
Previously stored PBMC samples were thawed the day of staining. Viability was evaluated by Zombie-Aqua VioBlue dye staining. Cells were washed with centrifugation with roomtemperature phosphate-buffered saline (PBS). After resuspending cells in PBS, flow antibodies were added (Supplementary Table S1). Samples were incubated at room temperature for 20 minutes in a dark environment. After a second wash with room-temperature PBS, cells were analyzed using an AttuneX machine. Postacquisition analysis was performed using FlowJo software.

Statistical analysis
Descriptive statistics (median and range, or frequency and percentage) were used to summarize patient demographics and disease characteristics. Wilcoxon rank-sum tests, chi-square tests or Fisher's exact tests were used to compare distributions of characteristics between non-progressors (NP) patients and patients with IP. The primary clinical outcomes evaluated were PFS and OS from ASCT. PFS was defined as the time from ASCT until either disease progression or death, while OS was defined as the time from ASCT until death or last follow-up. Poisson regression models with robust variance were used to evaluate the associations between patients' characteristics and IP. This analytic approach provides an unbiased estimate of the relative risk when the outcome is common (greater than 10%) (17). Cox proportional regression models were used for OS and PFS analysis. For each of the models above, univariable analysis (UVA) models were conducted first to evaluate the associations between individual risk factors and the study outcome. Factors with P values < 0.10 were further evaluated in a multivariable analysis (MVA) model to estimate its independent effect. Maintenance therapy was evaluated as a time dependent covariate in the model. Stata 16 (College Station, Texas) was used for all analyses and all tests were two-sided with significance level at 0.05. Immunophenotypic analysis was performed on either the percentage of specific cellular populations or the Mean Fluorescence Index (MFI). One-way ANOVA with Bonferroni's multiple comparisons analysis was used to compared immunophenotypic data from healthy donors (HD), patients with IP, or NP.
Prevalence of interval progression and its role in MM disease response pre-and post-ASCT One hundred and six (26%) patients experienced IP based on our definition criteria, i.e. a 25% increase of the index protein between the end of induction therapy and Day -2 of ASCT. The median time between diagnosis and ASCT was 159 days in nonprogressor (NP) patients (range: 84-361 days) and 177 days in IP patients (range: 86-359, P = 0.007) and the median time between the end of induction and ASCT was 36 days in NP patients (range: 12-90 days), and 43 days in IP patients (range: 13-83 days, P = 0.002).
No imbalance was noted in terms of dose of conditioning regimen with ASCT or maintenance therapy between the IP and NP groups.
The extent of disease throughout induction and consolidation therapy was monitored using response classifications developed by the IMWG working group ( Figure 1A and Supplementary Table S2).
The overall median follow-up among those alive was 6.2 years from ASCT, 6.1 years for NP and 6.3 years for patients with IP, respectively. Kaplan-Meier survival analysis for PFS and OS from ASCT can be found in Figures 2A, B. IP was associated with inferior survival outcomes mainly in PFS (P = 0.022 and P = 0.2 for PFS and OS, respectively). Five-year PFS rates were 44.4% (95% CI: 38.5%-50.2%) and 38.1% (95% CI: 28.6%-47.4%) in the NP and IP groups, with median PFS of 4.5 (95% CI: 4.0-5.1 years) and 3.0 (95% CI: 2.3-4.2) years, respectively. Five-year OS rates were 72.1% (95% CI: 66.4%-76.9%) in the NP group and 67.5% (95% CI: 57.2%-75.9%) in the IP group, with median OS of 9. Induction with bortezomib, lenalidomide, dexamethasone and its relationship with IP and outcomes To exclude that the above analysis may simply be reflective of suboptimal induction, we performed a separate analysis on 165 patients who received triplet-based induction therapy with VRD. The patient characteristics of this group were similar to the full cohort of patients (Supplementary Table S6 In this cohort of patients, inferior PFS and OS from ASCT were associated with IP (P = 0.001, and P = 0.03, Figures 3C, D). Five-year PFS rates were 44.4% (95% CI: 34.9%-53.4%) and 25.7% (95% CI: 12.7%-40.9%) in the NP and IP groups, with median PFS of 4.6 (95% CI: 4.0-5.6 years) and 2.5 (95% CI: 1.4-3.8) years, respectively. Five-year OS rates were 75.2% (95% CI: 66.0%-82.3%) in the NP group and 69.5% (95% CI: 50.3%-82.4%) in the IP group, with median OS of not reached (95% CI: 7.6-not reached) and 5.7 (95% CI: 5.  Table S9). This group of patients was representative of the cohort of all patients showing similar overall responses and inferior PFS from ASCT as in the full cohort (Supplementary Figures S2A, B).
We then evaluated NK cell subsets in healthy donors and in the same two groups of patients as above. The different cellular subsets were gated as previously described (15). Total NK cells, defined as CD3 -CD56 + cells, were distinguished by the relative expression of CD56 surface marker as either CD56 bright or CD56 dim cells. CD56 bright NK cells have low cytotoxicity activity, while CD56 dim NK cells are usually CD16 + and have more cytotoxic activity, representing an important source of NK-cell mediated antitumoral immunity. Together with CD16, DNAX accessory molecule 1 (DNAM1 or CD226), CD69, and NKG2D induce NK cell activation and enhance tumor lysis ( Figure 5A). Patients with IP  Figure 5D). The percentage of CD56 dim CD16 + NK cells is lower in both IP and NP patients compared with HD (P <0.0001 and P <0.0001, respectively). Despite no statistically difference in the percentage of CD56 dim CD16 + NK cells between NP and IP patients (P = 0.99), Figure 5E, there was a lower percentage of CD56 dim CD16 + NKG2D + cells (MSCP: 15.31% in IP versus 27.38% in NP, P = 0.046; HD: 41.0%; ANOVA Summary P = 0.0012), CD56 dim CD16 + CD226 + cells (MSCP: 16.59% in IP versus 31.54% in NP, P = 0.05; HD: 61.74%; ANOVA Summary P < 0.0001), and CD56 dim CD16 + CD69 + cells (MSCP: 11.06% IP versus 28.69% NP, P = 0.0091; HD: 51.83%; ANOVA Summary P < 0.0001) in patients with IP compared with NP ( Figure 5F). All NK cell activation markers were lower in MM patients than in HD.

Discussion
To produce a deep, sustained remission, transplant-eligible patients with MM receive six to eight cycles of induction therapy followed by melphalan conditioning therapy and ASCT, a strategy which ensures longer PFS compared to delayed transplant (3,18,19). By the nature of the treatment timeline, there is at least a month-long gap (median: 38 days in our full cohort; median: 35 days in patients treated with VRD induction) between the end of induction therapy and ASCT, during which patients are not receiving any disease-directed therapy.
CR or VGPR post-HDT/ASCT are known to be associated with improved survival outcomes (4-7), while the role of CR or VGPR post-induction or pre-ASCT is more controversial. Older studies using chemotherapeutic induction therapy demonstrated that patients who did not achieve CR post-induction still obtained a survival benefit from HDT/ASCT especially in PFS (8), while newer studies have mixed outcomes. Some suggest that depth of response at the end of induction or pre-ASCT still matters, recommending continuing induction therapy if CR is not obtained (20), while others focus on intensifying post-ASCT therapies with consolidation if no CR or MRD negativity is noted (21). This paper is the first to analyze long-term outcomes of patients who experienced progression of disease during the time gap between induction therapy and day of ASCT, an event not uncommon, as it occurred in a quarter of the patients of our database. In the entire cohort, IP was significantly associated with shorter PFS in the univariable analysis (HR = 1.37, P = 0.022) but not in the multivariable analysis (HR = 1.14, P = 0.44). However, analyzing only patients who received VRD as induction, progression-free survival (PFS) remained inferior in both the univariable (HR = 2.02; P = 0.002) and the multivariable analyses (HR = 1.96; P = 0.01). Our data also demonstrate that despite the clear short-term benefit of ASCT in patients with IP, they also tended to progress more commonly within the first 12 months from ASCT, a known poor prognostic feature in MM (22).
The presence of IP is also of additional significance because the collection of stem cells occurs during this time. A recent, prospective study showed MM cells in 40% of the collected stem cells, with the frequency of contamination and re-infusion directly correlating with the post-induction response and conferring a 2fold risk increase in not achieving or delaying CR post-HDT/ ASCT (23). Immune cell dysfunction in patients with MM is common. ASCT tends to accelerate MM-associated T cell defects, with patients with high expression of LAG-3 + T cells having a shorter response to ASCT (24). In our study, we investigated T and NK cell phenotypes in samples obtained just prior ASCT in patients who were not receiving MM-directed therapy; therefore, the influence of anti-MM drugs on immune cells likely attenuated. We observed an increase in CD3 + CD8 + CD57 + CD28 -, CD3 + CD4 + LAG-3 + and CD3 + CD4 + PD-1 + T cells in patients with IP. Despite their efficacy in solid tumors, anti-PD-1 antibodies especially in combination with immunomodulatory drugs, were associated with adverse outcomes and toxicity in MM (25,26). LAG-3 is another suitable target, with LAG-3 inhibitors increasing T cell function in in vitro studies, while currently being investigated in clinical trials (13). Similarly, reduced percentages of CD69 + , CD226 + , and NKG2D + activated NK cells in both the CD56 bright and CD56 dim subpopulations were noted in patients with IP. Even though we do not have functional data on NK cells, the reduction of activated receptors would likely limit recognition of MM cells by NK cells, compromising anti-tumoral immunity in patients with rapidly growing disease (27).
This study has some limitations. Firstly, it is a single center retrospective study not performed in a controlled clinical setting, and as such, patients received a variable number of induction cycles as well as different regimens. As the trials demonstrating triplet therapy superiority were published more recently, patients who received older doublet regimens were included to permit analysis of long-term survival outcomes. However, 40.4% of patients received the now preferred triplet induction therapy and the analysis in this group confirmed reduced PFS from ASCT. Additionally, as this was the first project to study the progression of disease between end of induction and day of transplant, the definition of IP was arbitrarily set by this group, based on the IMWG definition of a 25% increase in M protein constituting progressive disease, in patients with stable kidney function and at least 100 mg/dL of free light chains or 0.1 g/dL of M-protein. Using a different percentage cutoff may yield variable survival results and may represent a future avenue of study. Another limitation is the relatively small number of peripheral blood samples available to study, which is though in line with previous studies (14,15,24). While the number of samples is limited, these samples are representative of the full cohort. Moreover, matched PB samples at MM diagnosis were not available, which could have been informative on the initial presentation of immune dysfunction in these patients. Nor were matched PB samples at first relapse obtained, which could have revealed data on clonal evolution or persistence of the immune composition. In conclusion, we report that patients treated with VRD who experienced IP in the interval between induction therapy and ASCT have inferior PFS even in the MVA analysis, supporting the importance of achieving deep responses before proceeding to ASCT or other consolidation therapies to obtain PFS benefits. The management of patients with IP remains uncertain as it is unclear if they are destined to suboptimal outcomes independent of therapy or they can be salvaged with additional cycles of conventional therapy followed by ASCT or experimental strategies to modify their immune profiling. This study also confirms the fact that abnormal immune cell composition occurs early during the disease course and can potentially compromise future immunotherapeutic approaches. Therefore, early introduction of specific immunotherapy intervention, such as LAG-3 inhibition, might benefit patients with rapid disease progression or with an aggressive disease course.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by Ohio State University Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Author contributions
All authors contributed to the article and approved the submitted version. AB performed research, collected, and analyzed data. QZ performed statistical analysis. RK and JR performed flow cytometry analysis of the peripheral blood of patients with MM and healthy individuals, NS collected retrospective MM patient data, NB, SD, AK, EU, AR, and DB consented patients and provided critical review of manuscript. FC performed and supervised data collection and analysis, wrote the IRB protocol and manuscript, supervised the study, and obtained funding.